Detection of mutations in the dystrophin gene via automated DHPLC screening and direct sequencing

Abstract

Background: Currently molecular diagnostic laboratories focus only on the identification of large deletion and duplication mutations (spanning one exon or more) for Duchenne Muscular Dystrophy (DMD) yielding 65% of causative mutations. These mutations are detected by an existing set of multiplexed polymerase chain reaction (PCR) primer pairs. Due to the large size of the dystrophin gene (79 exons), finding point mutations (substitutions, deletions or insertions of one or several nucleotides) has been prohibitively expensive and laborious. The aim of this project was to develop an effective and convenient method of finding all, or most, mutations in the dystrophin gene with only a moderate increase in cost.

Results: Using denaturing high performance liquid chromatography (DHPLC) screening and direct sequencing, 86 PCR amplicons of genomic DNA from the dystrophin gene were screened for mutations in eight patients diagnosed with DMD who had tested negative for large DNA rearragements. Mutations likely to be disease-causative were found in six of the eight patients. All 86 amplicons from the two patients in whom no likely disease-causative mutations were found were completely sequenced and only polymorphisms were found.

Conclusions: We have shown that it is now feasible for clinical laboratories to begin testing for both point mutations and large deletions/duplications in the dystrophin gene. The detection rate will rise from 65% to greater than 92% with only a moderate increase in cost.

Figure 1

Electropherograms of dystrophin exon 39 sequence from Patients 01 and 01S UM-39F: Unaffected Male-Exon39 Forward UM-39R: Unaffected Male-Exon39 Reverse 01: Patient 01 01S: sister of patient 01

Figure 2

DHPLC chromatograms of unaffected male vs. patient a: patient 06 exon 59 A->G bp 9018 b: patient 08 exon 67 G->A bp 10015+5 c: patient 06 exon 66 C->T bp 9857+15

Figure 3

DHPLC chromatograms of unaffected male vs. patient a: patients 01 and 01s exon 39 C-> T bp 5762 b: patient 02 exon 41 del 6056–6066 c: patient 04 exon 06 G->T bp 738+1 d: patient 07 exon 23 C->T bp 3359

Figure 4

DHPLC chromatograms for variation negative condition and standards a: Typical chromatograms as seen when no variation exists between unaffected control and patients b: Mutation standard c: Size standard